When they investigate biological systems, molecular biologists and chemists time and again meet with the necessity of having to covalently bind two molecular units with each other, e.g. an oligosaccharide with a peptide, a reporter molecule with a biopolymer, two biopolymers with each other or a low-molecular therapeutic agent with a biopolymer. Usually, all of said compounds have a number of chemical functions whose respective reaction behaviour has to be observed under the conditions of the ligation reaction. Hence it follows that the chemoselective ligation reactions should have a clear reaction course without the other existing chemical functions or groups being attacked or actively interfering with the reaction event. This can only be realized if two functional groups reacting selectively with each other are involved in the ligation step. Furthermore, it would be desirable that such a ligation reaction can proceed without the use of protective groups in every environment and at a pH adapted to the respective biopolymer.
One of the few chemical reactions which can fully comply with all of these conditions is the cycloaddition, either of the 4+2 type, which is known as the Diels Alder reaction (FIG. 1; J. Sauer, 1966, Angew. Chem. 78, 233), or of the 3+2 type, which is known as 1,3 dipolar cycloaddition. The sharpless ligation was developed on the basis of this reaction (lit). A method also further developed in the past few years is the Staudinger ligation (Review: Angew. Chem. 2004, 116, 3168-3178).
DE-A-100 41 221.1 shows the application of the classical Diels Alder reaction as a ligation reaction, the diene being provided with electron-donating substituents and the dienophile being provided with electron-attracting substituents. In this connection, furan and its derivatives were used as dienes, and substituted maleinimides were used as dienophiles. This system was selected due to the simple accessibility of the respective components and the simple chemistry thereof. Many furans can easily be produced from saccharides and are available in major amounts. Like many chemical reactions, the Diels Alder reaction (hereinafter referred to as “DAR”) can be reversed, above all at elevated temperatures. This reversibility is particularly developed in the furan/maleinimide system, which is caused by the high reactivity of the maleinimides for nucleophilic additions. This can readily be read off the use of maleinimides for labeling peptides or the linkage thereof. In this connection, the thiol group of the protein adds to the double bond of the maleinimide in a very rapid, irreversible reaction. Already the small amounts of maleinimide which are present by the back reaction of the DAR in the equilibrium are trapped by such an addition thus shifting the equilibrium towards the starting substances. This is a real drawback of the DAR since this significantly minimizes the yield of the desired product.